| Primary nephrotic syndrome ( PNS) is one of the common renal diseases in children, the pathogenesis of which is unclear. There is the evidence suggests that the proteinuria of NS is associated to the increasing expression of the interluikin -8(IL -8) gene and protein.There is trace of IL - 8 in the peripheral blood, as the most active chemokine. Garin et al. pointed out IL - 8 might induce the proteinuria of PNS, which is the higher permeability of the glomerular basement membrane ( GBM ) to serum albumin due to a defective glomerular negative charge - selective barrier caused by the increasing the ca-tabolism of sulfide in GBM through animal experiment, methylprednisolone pulse therapy of PNS is better than the routine oral prednisone therapy because of the shorter time to remission and less side effect. And there is no report about change of the serology and gene expression of IL - 8 in short time after the methylprednisolone pulse therapy.The purpose of the work was to evaluate serum concentration and mRNA expression of IL - 8 before and after the methylprednisolone pulse therapy (MPT) of PNS, using reverse transcriptase PCR (RT -PCR)and enzyme -linked immunosorbent assay(ELISA).Experimental materialsObjects for serum IL - 8 level Patients group includes Before treatment group ( Group B ) and After treatment group ( Group A ): Thirty children with diagnosed PNS from Dec 2000 to Oct 2001 were enrolled in our study. They all hadn't have the glucocoticoid therapy, or in less 3 months. Of the cases ,24 were male and 6 were female. The median age was 8. 5 years ( age range from 1.5 to 14 ). Health control group was made up of eighteen health children for health examination. 13 were male and 5 were female. The average age is 8(2 ~ 14) .Objects for PBMC DL - 8 mRNA expression Patients group (Group B and Group A) ;Twenty children patients were selected from above thirty patients,so as eight control group children randomly.Main reagents and instruments Human IL - 8 ELISA kit was purchased from Jingmei corporation Shenzhen, Trizol reagent and First strand cDNA synthesis kit all from GIBCO BRL USA; EL -309(U. S. A) auto enzyme label meter, PTC -200 MJ RESEARCH USA PCR amplier, GIS 700D tianon gel scan and analysis system.MethodsSamples:2ml vein blood and 5 ml heparin anticoagulant blood were collected before and after MPT respectively and preserved at -70℃ before use.The serum protein level of IL - 8 was measured through ELISA according to the manufacturers instructions. And the concentration was obtained after drawing the standard curve.RT - PCR: The isolation of the total RNA and the synthesis of single - stranded cDNA were all performed according to the manual. And the PCR amplification was accomplished on the PTC -200 under the condition ( 94℃ degeneration for 5 mins;then 35 circulations at the sequence of 94℃ 30 seconds,55℃ 1 minute, 12℃ 2 mins;72℃ extension 10 mins at the last).Rand sum test statistical analysis was adopted for dealing with the data.ResultsComparison of the serum level of IL - 8 in before therapy group and after therapy group showed significant difference ( 7. 14 -352.08 pg/ml vs. 4.27 -77.86 pg/ml, u =4.26,P <0. 01) .And the serum level in before therapy group was increasing obviously comparison to the level in control group(5.46 -33.31 pg/ml, u=4.53 P<0.01). Then the comparison of after therapy group and control group showed significant difference also(u =2.13 P <0.01).Comparison of the mRNA expression of IL - 8 in before therapy group and after therapy group showed significant difference (0. 776 ~ 0.95 vs. 0 - 0.754, u = 3. 902 P < 0.01) . We also found the obvious difference between the before therapy group and control group through the clearer strap in the before therapy group by the electrophoregram.DiscussionThere is a close relationship between the IL - 8 and proteinuria of PNS patients. Garin suggested that there was a cytokine in supernatantfrom peripheral blood mononuclear cell(PBMC) cultures of PNS patients, and the s... |
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